Currently, there is no way to regenerate limbs that are lost to injuries or diseases. Our long term goal is to understand the role of the nail epithelim during digit tip regeneration and propose a novel strategy to promote regeneration of the digit/limb. The mammalian digit tip including its bone can regenerate upon amputation. Following amputation of the digit tip, the nail epithelium regrows together with the underlying undifferentiated mesenchymal blastema cells that regenerates the digit bone. Regeneration occurs only in areas associated with the nail and it is unknown why this regenerative limitation exists. We recently identified and discovered nail epithelial stem cells (NSCs) undergo Wnt-dependent nail differentiation. Remarkably, Wnt activation in the nail epithelium is required not only for nail regeneration but also for mesenchymal blastema growth that leads to digit bone regeneration upon digit tip amputation. When the digit is amputated proximal to the Wnt-active nail progenitors, Wnt activation does not occur in epithelial cells in the wound area and the nail/digit fails to regenerate. Nevertheless, forced Wnt activation in epithelial cells including NSCs can overcome this limitation. We hypothesize that Wnt-active nail epithelium holds a lineage specific function to promote digit regeneration by emitting paracrine signals. Aim1: We found that FGF2 is expressed in nail epithelial cells in a Wnt-dependent manner after amputation and that FGF2 promotes blastema proliferation through FGFR1 signaling in vitro. We will test whether FGFR1 signaling is essential for blastema growth in vivo, by genetically deleting fgfr1 in blastema cells. We will also test if overexpression of fgf2 in epithelial cells cn overcome the proximal limitation in regeneration. These experiments will be the first to dissect the role of a molecular pathway in digit tip regeneration by separately targeting the epithelial an mesenchymal cells. Aim2: [While the essential role of Wnt-active nail epithelial cells in inducing digit regeneration has been demonstrated, it remains unclear whether Wnt-active NSC-derived epithelial cells hold a unique lineage-dependent function to promote regeneration. We will thus test whether Wnt-activation in non-NSC-derived epithelial cells (i.e. epithelial cells proximal to nail epithelium) can similarly function to promote regeneration as those derived from the NSC lineage. Non-NSC-derived epithelial cells with stabilized -catenin lack expression of TCF/LEF1 family members, transcription factors essential for Wnt induced transcription, in contrast to nail epithelial cells that display both -catenin stabilization and express TCF1. We plan to induce Wnt signal activation in non- NSC lineage by genetically expressing TCF1 in combination with stabilized -catenin following amputations proximal to the NSC niche and examine whether this is sufficient to induce digit regeneration. Additionally, we will transplant NSCs and normal skin keratinocytes that constitutively activate Wnt signaling underneath the wound epidermis. We will test if this cell therapy can induce digit regeneration following proximal amputation.] These experiments will create a foundation to exploit NSCs in regenerative medicine for treating amputees.